Three cell surface antigens expressed on melanomas are now available in purified form, the gangliosides GD2, GD3 and GM2. All three serve as differentiation antigens for different subsets of cells of neuroectodermal lineage. Both GD2 and GM2 are known to be potentially immunogenic in man as they have been identified by a variety of human sera. The clinical relevance of GD3 has been demonstrated by inflammatory reactions and major clinical responses in 3 melanoma patients treated with anti-GD3 monoclonal antibody (R24). We have however, been unable to develop consistently immunogenic vaccines in man or in the mouse using whole cells or cell lysates expressing these gangliosides. Consequently, we have explored the effect of purified GM2 and GD2 vaccines in the mouse and have identified approaches that consistently induce an antibody response. The success of these murine trials has encouraged us to proceed with similar trials in Stage II melanoma patients. In our initial 3 trials, none of 6 patients immunized with GM2 alone produced antibody but 5 of 11 patients immunized with GM2 plus BCG or GM2 plus Salmonella minn. mutant R595 have produced antibody reactive with GM2 (median titer 320). These sera mediate complement dependent cytotoxicity on human melanoma and astrocytoma cells with human complement. ITLC confirmed that they react exclusively with GM2. The approaches proposed here are (and will continue to be) based on the results of these ongoing studies in the mouse. In intitial trials we will change the dose, schedule and route of administration of GM2 plus BCG or R595 or both to further increase the anti GM2 response rate. Subsequent trials will test other vaccines such as liposomes containing GM2 and GM2 covalently attached to BSA or KLH. Stage II melanoma patients will be vaccinated after, or before and after, lymphadenectomy in small groups and their serological reactivity and delayed hypersensitivity reactions to the relevant gangliosides tested. The immunized lymph nodes removed at surgery will be used to produce human monoclonal antibodies. Approaches provoking a consistent serological or DTH responses will be used with GD2 and GD3, and subsequently with the three gangliosides together. Vaccines producing a consistent response in Stage II patients will be used in patients with measurable disease to gauge their effect on immune responses in the face of more advanced disease.